Sperm staining and sorting methods

ABSTRACT

A method of sex sorting sperm is disclosed. The sperm may be stained with a DNA selective fluorescent dye, which fluoresces when excited, a dead quenching dye, which selectively quenches fluorescence emitted by the DNA selective fluorescent dye within the membrane of compromised sperm, and a split enhancing dye. The stained sperm may then sorted into one or more gender enriched subpopulations of viable sperm.

This application claims priority to, and the benefit of, U.S.provisional application 61/541,438, filed on Sep. 30, 2011, and U.S.provisional application No. 61/541,451, filed on Sep. 30, 2011, eachapplication is hereby incorporated herein by reference.

TECHNICAL FIELD

Generally, this disclosure relates to staining and sorting methods, suchas Fluorescence Activated Cell Sorting (FACS), and more particularlythis disclosure relates to staining and sorting methods which improvesort parameters in flow cytometry.

BACKGROUND

Effective pre-selection of sex has been accomplished in many speciesfollowing the development of reliable methods for separating sperm intoenriched X-chromosome and/or Y-chromosome bearing subpopulations.Traditional sperm sorting methods often rely on FACS for the detectionof quantifiable differences in DNA content of X-chromosome bearing spermand Y-chromosome bearing sperm, such as through flow cytometry. Atypical step in these methods includes staining a sperm population witha DNA selective fluorescent dye that uniformly stains nuclear DNA.Hoechst 33342, sometimes referred to as Hoechst bisbenzimide 33342, isthe most widely utilized stain for this purpose because it can be usedin a sufficient quantity to differentiate small variations in nuclearDNA without exhibiting the toxicity of other fluorescent stains.

Sorted sperm quality and purities improved with the introduction of adead quenching dye for differentiating membrane compromised sperm fromthe population or populations of interest. Membrane compromised spermcan include dead and dying sperm, which may have fragmented anddisintegrating nuclear DNA that obscures the already narrow differencesin X-chromosome bearing sperm and Y-chromosome bearing sperm. Initially,propidium iodine was used for this purpose, but has been shown to betoxic to sperm. Later, FD&C red food dye No. 40,6-hydroxy-5-[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-naphthalenesulfonicacid (hereafter “red food dye No. 40” or “red 40”), was added to thesperm staining process as the dead quenching dye for quenching membranecompromised sperm and removing membrane compromised sperm from the sortanalysis.

When sex sorting sperm, ultimately a small variation in DNA content isquantified differentiating X-chromosome bearing sperm from Y-chromosomebearing sperm. In bovine, for example, Holsteins have about a 3.8%difference in DNA content, while Jersey bulls have about a 4.1%difference. Due to the inexact nature of stoichiometric DNA staining,these small differences can be difficult to differentiate. Thisdifficulty may be exacerbated by noise in the DNA staining complex,which can vary randomly in both X-chromosome bearing sperm andY-chromosome bearing sperm.

One previously unrealized problem that may exist in current deadquenching staining regiments may be an over quenching of some desiredsignal. This may occur with the presence of a dead quenching dye in asperm sample which does not associate with membrane compromised sperm,but instead remains in the sample quenching signals produced by membranecompromised sperm and non-membrane compromised sperm alike. It may alsobe that some dead quenching dye associates with healthy sperm cells. Forthese reasons, quenching in the sample may slightly quench the overallsignal. While this may be happening to a far extent as compared to thequenching of membrane compromised sperm, it can be relevant whenattempting to differentiate small differences in nuclear DNA content.

The sperm sorting process is damaging to cells which arenon-regenerative time critical cells. The staining step can be theespecially harmful. While Hoechst 33342 can be used in non-toxicconcentrations, sperm must be incubated at elevated temperatures andelevated pHs for sufficient Hoechst 33342 penetration with sufficientuniformity for analysis or sorting. Each of elevating sperm temperatureand elevating sperm pH may contribute to sperm damage. Therefore, a needexists for improvements to the staining process which reduce stain timesor reduce incubation temperatures and/or pHs. Additionally, manychemicals added during sperm processing negatively impact spermviability. This can be particularly true in sperm staining and sortingprocedures.

DISCLOSURE OF INVENTION

Certain embodiments of the claimed invention are summarized below. Theseembodiments are not intended to limit the scope of the claimedinvention, but rather serve as brief descriptions of possible forms ofthe invention. The invention may encompass a variety of forms whichdiffer from these summaries.

One embodiment relates to a method of gender enriching one or more spermsubpopulation utilizing a dead quenching dye and a split enhancing dye.Such a method can begin by obtaining a population of sperm whichincludes both X-chromosome bearing sperm and Y-chromosome bearing sperm.At least a portion of that sperm can be stained with a DNA selectivefluorescent dye which fluoresces when excited. The stained portion ofsperm may then be further stained with a dead quenching dye whichselectively quenches fluorescence emitted by the DNA selectivefluorescent dye within the membrane of compromised sperm. The stainedportion of sperm may additionally be stained with a split enhancing dyeand then sorted into one or more gender enriched subpopulations ofviable sperm.

Another embodiment relates to a method for gender enriching one or moresperm subpopulations with a dead quenching dye having a yellow ororangish color. Such a method can begin by obtaining a population ofsperm which includes both X-chromosome bearing sperm and Y-chromosomebearing sperm. At least a portion of that sperm can be stained with aDNA selective fluorescent dye which fluoresces when excited. The stainedportion of sperm may then be further stained with a dead quenching dyewhich selectively quenches fluorescence emitted by the DNA selectivefluorescent dye within the membrane of compromised sperm. The deadquenching dye may be yellow, orange, or orangish red in appearance.Finally, the stained sperm may be sorted into one or more genderenriched subpopulations of viable sperm.

Still another embodiment relates to a sex sorted sperm suspension in theform of either an intermediate product in the sorting process, or as anartificial inseminate. The sex sorted sperm suspension can includeviable, sex sorted sperm including an associated DNA selectivefluorescent dye. The suspension may additionally include a mediumsupporting sperm viability, as well as, a first dye and a second dye.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a representation of a jet-in-air flow cytometer usedin certain embodiments described herein.

FIG. 2 illustrates an excitation curve for Hoechst 33342 as compared toabsorption curves for several dyes.

FIG. 3 illustrates bivariate histograms from a flow cytometer sortingsperm with different dead quenching dyes.

FIG. 4 illustrates an example of parameters measured from a univariateand bivariate plot in a flow cytometer.

FIG. 5 illustrates a graphical comparison of the quenching capacity ofvarious dyes as dead quenching dyes.

FIG. 6 illustrates a graphical comparison of the split quality producedwhen sorting with various dyes.

FIG. 7A illustrates a graphical comparison of the quenching capacity ofvarious dyes as dead quenching dyes.

FIG. 7B illustrates a graphical comparison of the split quality producedwhen sorting with various dyes.

FIG. 8 illustrates a graphical comparison of the quenching capacity ofvarious split enhancing dyes used in combination with a dead quenchingdye.

FIG. 9 illustrates a graphical representation of a split enhancingcapacity at various concentrations.

While the present invention may be embodied with various modificationsand alternative forms, specific embodiments are illustrated in thefigures and described herein by way of illustrative examples. It shouldbe understood the figures and detailed descriptions are not intended tolimit the scope of the invention to the particular form disclosed, butthat all modifications, alternatives, and equivalents falling within thespirit and scope of the claims are intended to be covered.

MODES FOR CARRYING OUT THE INVENTION

In certain aspects the present invention provides improved stainingprocedures with particular benefits in the sex selection or sex sortingof sperm. For example, certain aspects relate to improvements in sortingsperm by quenching undesirable noise arising from random signalvariations produced by undesired dead cells and reducing undesirablenon-specific noise arising from viable cells. Another aspect relates toan improved dead quenching dye which with an improved capacity to quenchsignals from dead sperm. These aspects may provide independent benefitsin sort speeds or improved sort purities, or may be combined tosynergistically further improve sorting. Alternatively, stainconcentrations or stain times and other harmful aspects of staining maybe reduced with the described improvements while maintaining acceptablesorting parameters, such as speed and purity, perhaps resulting inimproved motility and viability in the sorted sperm sample.

Existing staining protocols for sex sorting, or even bulk sorting, spermrely up the inclusion of F&DC red food dye No. 40 (“red food dye No. 40”or “red 40”) as a dead quenching dye. The maximal absorbance wavelengthsof red food dye No. 40 overlaps the maximal emissions wavelengths offluorescent dyes, including Hoechst 33342 when bound to nuclear orchromosomal DNA. Because red food dye No. 40 differentially permeatesmembrane compromised sperm cells and overlaps the emission spectra ofthe DNA selective fluorescent dye, FRET (florescence resonance energytransfer) between the light leaving the DNA-stain complex and the deadquenching dye reduces the overall detected intensity of the lightemitted from membrane compromised sperm. The quenched, or dampened,fluorescence from these cells provide fewer photons to the detectorsresulting in a distinctly lower signal. This distinctly lower signalresults in a noticeable separated subpopulation which allows theexclusion (“gating out”) of the membrane compromised sperm during thesorting procedure. Since membrane compromised sperm comprises largelynon-viable sperm, excluding these cells from the analysis results in anenriched sperm subpopulation with respect to viability in the sex sortedsubpopulation.

In addition to the desired signal produced by chromosomal or nuclear DNAduring sorting, non-specific florescence can be produced from viablesperm during the sorting process. Non-specific florescence may includeflorescence emissions not coming from the DNA-stain complex, such asauto-florescence from NADH and NADPH, stain that may have associatedwith mitochondrial DNA, stain that may have associated with RNA, and/orstain that may have associated with cellular lipophilic components.While the exact source of non-specific noise from viable sperm is notknown with certainty, it was appreciated by applicants for the firsttime that this noise may be, at least partially, at a differentwavelength that the signal of interest. Namely, non-specific noise may,at least partially, occur in the 520 nm to 600 nm wavelength range, orperhaps at about 520 nm. Certain embodiments of the present inventionprovide split enhancing dyes which are impedance matched to thisnon-specific noise. Such split enhancing dyes may help improve sortingsignals and sort purities by enhancing the distinction between two spermsubpopulations.

In this way, staining procedures may be improved to enhance splits byreducing the non-specific fluorescence of live sperm with the additionof split enhancing dyes impedance matched to that non-specificfluorescence. Since non-specific florescence is not correlated with thesex chromosome of sperm cells, if non-specific florescence has a high CV(coefficient of variation), then it may contribute a “noise signal” tothe CV of the populations of live X-chromosome bearing and Y-chromosomebearing sperm. Even if the non-specific florescence is very low (such as0.05%-0.25% of total florescence) if variation is high, it maycontribute significant variation, causing a reduction in the resolutionbetween the two closely related sex populations, resulting in what isgenerally called a “poor split”. The result of a poor split is a slowersorting process, or conversely a good split is deemed essential to afast sorting process.

In one aspect the invention provides a method of selecting genderenriched populations of sperm including the steps of: (1) obtaining apopulation of sperm; (2) staining the population of sperm with a DNAselective dye, a dead quenching dye, and, in some cases, a splitenhancing dye; and (3) sorting the stained population of sperm. Certainembodiments described herein relate to mixed dyes with enhance sortingproperties, while other embodiments relate to improved dead quenchingdyes that improve sort parameters, such as, sort speed and sort purity.

In the first step (1), a population of sperm is obtained. The populationof sperm can be obtained in the form of neat semen, extended sperm,frozen thawed sperm or in combinations thereof. The population of spermcan be obtained at the same location as the remaining steps, or can beextended in an appropriate sperm buffer for transport to a sortingfacility. The sperm can be maintained at room temperature, chilled, oreven frozen in an appropriate buffer for later use. The step ofobtaining sperm can include obtaining a frozen or chilled straw fromstorage, or even pooling frozen or extended sperm.

The population of sperm can originate from mammals, such as a non-humanmammals listed by Wilson, D. E. AND Reeder, D. M., Mammal Species of theWorld, Smithsonian Institution Press, (1993), the entire contents ofwhich are incorporated herein by reference.

In the second step (2), the population of sperm is stained in a processwhich includes a DNA selective fluorescent dye, a dead quenching dye andin some cases a split enhancing dye. The population of sperm can includeX-chromosome bearing sperm and Y-chromosome bearing sperm. Additionally,each of the X-chromosome bearing sperm and the Y-chromosome bearingsperm can include viable sperm and nonviable sperm. Viable sperm can beconsidered sperm with intact membranes while nonviable sperm can beconsidered sperm with compromised membranes. Viable sperm, in theappropriate dosage, will generally be capable of achieving fertilizationin an artificial insemination, while nonviable sperm, or membranecompromised sperm, will be incapable of achieving fertilization in anartificial insemination or will have a greatly reduced ability to do so.However, some sperm capable of fertilization may have compromisedmembranes, and some sperm with intact membranes may be incapable offertilization. The staining can take place in two distinct steps. In thefirst step, at least a portion of the population of sperm is incubatedwith a first staining buffer and a DNA selective fluorescent dye. Thepurpose of the first step is to stoichiometrically stain the DNA contentof each cell in the sperm population. Hoechst 33342 is described in U.S.Pat. No. 5,135,759, for this purpose. However, other UV excitable dyes,as well as visible light excitable dyes, fluorescent polyamides,fluorescent nucleotide sequences, and sex specific antibodies could alsobe used for this purpose.

Sperm in its natural state is often not readily permeable to such dyes.In order to produce a uniform staining, the first step of staining caninclude incubating at least a portion of the sperm population at anelevated temperature in a first staining buffer at an elevated pH inaddition to the dye. Examples of appropriate first staining buffers canbe a TALP, TES-TRIS, TRIS citrate, sodium citrate, or a HEPES basedmedium, each described in WO2005/095960, incorporated herein byreference. An exemplary modified TALP described in WO2001/37655,incorporated herein by reference, is illustrated in Table 1.

TABLE 1 Modified TALP buffer Ingredient Concentration NaCl 95.0 mM KCl3.0 mM NaHPO₄ 0.3 mM NaHCO₃ 10.0 mM MgCL₂ 6H₂O 0.4 mM Na Pyruvate 2.0 mMGlucose 5.0 mM Na Lactate 25.0 mM HEPES 40.0 mM bovine serum albumin 3.0mg/ml

As one example, the population of sperm, or a portion of the populationof sperm, could be diluted with the first buffer to between 640×10⁶ and40×10⁶ sperm/ml, to between about 320×10⁶ and 80×10⁶ sperm/ml, or toabout 160×10⁶ sperm/ml in the first buffer. The DNA selective florescentdye can be added to the sperm suspended in the first buffer in aconcentration of between about 10 μM and 200 μM; between about 20 μM and100 μM, or between about 30 μM and 70 μM. The pH of the first buffer canbe between about 6.8 and 7.9; about 7.1 and 7.6; or at about 7.4 inorder to help ensure a uniform staining of nuclear DNA. Those ofordinary skill in the art will appreciate the pH can be elevated withthe addition of NaOH and dropped with the addition of HCl.

The population of sperm can be incubated between 30-39° C., betweenabout 32-37° C., or at about 34° C. The period of incubation can rangebetween about 20 minutes and about an hour and a half, between about 30minutes and about 75 minutes, or for about 45 minutes to about 60minutes. As one example, the population of sperm can be incubated forabout 45 minutes at 34° C. Even within a single species, spermconcentration and pH and other factors affecting stainability can varyfrom animal to animal. Those of ordinary skill in the art can appreciateminor variations for incubating sperm between species and even betweenbreeds or animals of the same breed to achieve uniform staining withoutover staining a population of sperm.

At the end of the incubation period, when the DNA selective fluorescentdye has sufficiently saturated the population of sperm to differentiateX-chromosome bearing sperm from Y chromosome bearing sperm, a secondstep of staining can include a further dilution in a second stainingbuffer that contains one or more additional dyes. The one or moreadditional dyes can include a single dye for the purpose of permeatingand quenching signals from membrane compromised sperm cells, as well as,for reducing signal noise. The one or more additional dyes can alsoinclude the combination of a dead quenching dye and a split enhancingdye. The term “dead quenching dye” can be understood to include dyeswhich differentially associate with membrane compromised sperm. It maybe that these dyes enter membrane compromised sperm cells more easilybecause the membranes are breaking down or otherwise increasinglyporous, but it may also be that dead quenching dyes readily enter allsperm cells and that healthy sperm cells act to pump dead quenching dyesout faster than membrane compromised sperm. In either case, the spermcells with which the dead quenching dyes associate includes a largeportion of dead and dying sperm cells, although not necessarily all deadand dying sperm cells.

In an alternative embodiment, the dead quenching dye and/or the splitenhancing dye may be introduced with the first staining buffer havingthe DNA selective dye, or in both the first staining buffer and thesecond staining buffer.

The second buffer can be applied in an equal volume to half the currentsperm concentration. Similarly, the volume of the second buffer can beselected to achieve a desired final concentration of sperm for furtherprocessing, such as between about 320×10⁶ and 20×10⁶ sperm/ml, betweenabout 160×10⁶ and 40×10⁶ sperm/ml, or about 80×10⁶ sperm/ml. The secondbuffer can have a pH coordinated to bring the overall pH back to anideal pH for sperm. In the case of bovine, where the pH has been takento 7.4 with the first buffer, the second buffer can be applied at 5.5 inorder to bring the final pH back to 6.8. The specific pH can vary fromspecies to species, but that those of skill in the art can determineappropriate sperm pH and coordinate the second buffer to achieve aparticular desired pH. In another embodiment, the second buffer can havethe same pH as the first buffer.

The second buffer can be selected as a buffer similar to the firstbuffer. The pH of the second buffer can be reduced with the addition ofHCl. As one example, the modified TALP can be used with the addition ofa dead quenching dye, a split enhancing dye and with the addition of eggyolk, for example 4% egg yolk.

The dead quenching dye can selectively quench the fluorescence ofunwanted cells. To achieve this, the dye must enter unwanted cells andefficiently absorb the fluorescent emission of the DNA selective dye. Inthe case of sperm sorting, membrane compromised sperm are permeable tomany dyes. Red food dye No. 40 was previously used for this purpose.However, improvements in quenching efficiency are possible where thedead quenching dye is impedance matched more closely to the excitationof the DNA selective fluorescent dye. In the case of sperm, a common DNAselective fluorescent dye is Hoechst 33342, described above, which isexpected to fluoresce at a maximum emission at about 483 nm in theemission spectra. Improved quenching dyes may therefore be impedancematched to this emission value when they have corresponding absorptionmaximums at near the same value. Table 2 below illustrates several fooddyes and their expected absorption maxima in water. Additionally, FIG. 2provides a visual representation of the emission intensity of Hoechst33342, when excited by a UV laser in comparison to the absorptionspectra of yellow food dye No. 5 (Y5), yellow food dye No. 6 (Y6), redfood dye No. 40 (R40), red food dye No. 3 (R3), red food dye No. 4 (R4),phenol red, and violet.

TABLE 2 Expected Absorption Maxima (nm) of FD&C Dyes and Phenol Red DyeMaxima^(a)(nm) Solvent Absorption Yellow 5 water 422 Phenol Red water440 560 Yellow 6 water 480 Red 40 water 505 Red 2 water 520 Red 4(natural) water 520 555 Red 3 water 530 Blue 2 water 610 Green 3 water625 Blue 1 water 627 ^(a)The maxima will vary slightly due to variationsin solvent.

Exemplary dead quenching dyes can be those having lower absorptionmaxima than red food dye No. 40. Such dyes may be visually characterizedas yellow, orange, or orangish-red in color and may have absorptionmaxima in the range of between about 420 nm and about 500 nm. Exemplarydyes can include yellow dyes, such as FD&C yellow food dye no. 6,Disodium 6-hydroxy-5-[(4-sulfophenyl)azo]-2-naphthalenesulfononate,(hereafter “yellow food dye no. 6” or “yellow 6”); and FD&C yellow fooddye No. 5, Trisodium(4E)-5-oxo-1-(4-sulfonatophenyl)-4-[(4-sulfonatophenyl)hydrazono]-3-pyrazolecarboxylate,(hereafter “yellow food dye no. 5” or “yellow 5”). Both food dyesdemonstrate an ability to quench membrane compromised sperm cells in thesex sorting process.

The dead quenching dye is selected for its ability to permeate membranecompromised sperm cells and to dampen the fluorescence produced by theDNA selective fluorescent dye in those cells. Some orange and yellowfood dyes demonstrate an improved ability to differentiate membranecompromised sperm from healthier sperm. For example, yellow food dye No.6 tends to exhibit superior quenching to previous dead quenching dyes.Yellow food dye No. 6 may permeate membrane compromised sperm moreeffectively, or may possess a greater ability to absorb photons. At thesame time some yellow dyes demonstrate less dampening to the overallsignal. These benefits allow for less DNA selective fluorescent dye withthe first staining buffer and less yellow dye with the second stainingbuffer, as compared the amount of red food dye No. 40 previously used.

As dead quenching dyes, yellow food dye No. 6 can be used in aconcentration of greater than 12.5 μM, between 12.5 μM and 250 μM, orbetween 20 μM and 125 μM and 500 μM. Yellow food dye No. 5 can be usedin concentrations greater than 125 μM, or between about 125 μM and 500μM.

Exemplary split enhancing dyes can be red dyes other than red food dyeno. 40, or perhaps even redish and violet dyes. The local absorptionmaximum wavelength of split enhancing dyes may be greater than that ofred food dye No. 40, namely greater than 505 nm. The local absorptionmaximum wavelength may be greater than about 520 nm. Alternatively, thesplit enhancing dye can have a local absorption maximum wavelengthbetween about 520 nm and about 600 nm. Exemplary dyes can include phenolred, sometimes referred to as phenolsulfonphthalein or PSP; natural redfood dye No. 4, known as carmine or natural red 4 (hereafter “carmine”or “red food dye No. 4”); and FD&C red food dye No. 3,2-(6-Hydroxy-2,4,5,7-tetraiodo-3-oxo-xanthen-9-yl)benzoic acid,(hereafter “red food dye No. 3” or “red 3”). The examples belowdemonstrate phenol red and red food dye No. 4 as improving the splitquality at a number of concentrations. Red food dye No. 3 also improvessorting at some concentrations. With reference to Table 2, Phenol red isbelieved to have a maximum absorption at about 560 nm to 570 nm (in thered molecular form), red food dye No. 4 is believed to have localmaximum absorption wavelengths at 520 nm and 555 nm, while red food dyeNo. 3 has an absorption maximum at about 530 nm. Dyes having similarmolecular weights and absorption maxima are expected also provideimprovements in sorting. For example, dyes having local absorptionmaxima in the range of about 520 nm to about 600 nm, or about 530 nm toabout 580 nm are expected to also provide improvements in the signal tonoise ratio. Possible autofluorescence and other unintendedfluorescence, such as possibly Hoechst 33342 bound to mitochondrial DNAmay to emit in the spectral range of about 520-600 nm. Therefore, byimpedance matching a split enhancing dye to have local absorption maximaat those wavelengths, noise can be reduced in the overall signal.

In some embodiments, the split enhancing dye can be used in addition toa dead quenching dye, while in other embodiments the split enhancing dyecan double as a dead quenching dye. As one example, phenol red, red fooddye No. 4, or red food dye No. 3 can be used as split enhancing dyes incombination with yellow food dye No. 6 for quenching membranecompromised sperm. Each of phenol red, red food dye No. 4, and red fooddye No. 3 have surprisingly been found to reduce noise and enhancesplits independent of dead quenching in sperm sorting.

Phenol red and red food dye No. 4 may be used as a split enhancing dyeat concentrations between 0.1 μM, and 1200 μM, and between 5 μM and 620μM, and between 15 μM and 125 μM. Red food dye No. 3 may be used as asplit enhancing dye in concentrations between about 25 μM and 600 μM,and 150 μM and 600 μM.

In some embodiments split enhancing dyes may be used for quenchingmembrane compromised sperm, although they tend to be less efficient atdoing so and require greater concentrations. For example, phenol red canbe used for quenching membrane compromised sperm in concentrationsbetween about 250 μM, and about 1250 μM.

In step (3) the stained population of sperm can be sorted, such as byflow cytometry. With reference to FIG. 1, a flow cytometer (10) is shownfor sorting stained sperm. The flow cytometer (10) includes a cellsource (12) supplying a sample for sorting. The sample includes thestained sperm, such as sperm stained with a DNA selective fluorescentdye and one or more other dyes. The stained sperm are deposited within anozzle (14) and introduced into a fluid stream (16) of sheath fluid(18). The sheath fluid (18) can be supplied by a sheath fluid source(20) so that as the cell source (12) supplies the sperm into the sheathfluid (18) they are concurrently fed through the nozzle (14). In thismanner the sheath fluid (18) forms a fluid stream coaxial to the samplehaving stained sperm. Since the various fluids are provided to the flowcytometer (10) at some pressure, they flow out of nozzle (14) and exitat the nozzle orifice (22). By providing an oscillator (24) which may bevery precisely controlled through an oscillator control (26), pressurewaves may be established within the nozzle (14) and transmitted to thefluids exiting the nozzle (14) at nozzle orifice (22). Since theoscillator (24) acts upon the sheath fluid (18), the fluid stream (16)exiting the nozzle orifice (22) eventually and regularly forms drops(28) at precise frequencies and velocities. Because the stained spermare surrounded by the fluid stream (16) or sheath fluid environment, thedrops (28) may contain within them individually isolated spermatozoa.

Since the drops (28) can contain individual spermatozoa, the flowcytometer can be used to sort sperm based upon individual cellcharacteristics. This is accomplished through a cell sensing system(30). The cell sensing system (30) includes at least a sensor (32)responsive to the cells contained within fluid stream (16). The cellsensing system (30) may cause an action depending upon the relativepresence or relative absence of a characteristic. Certaincharacteristics, such as the relative DNA content of sperm cells, can bedetected through excitation with an electromagnetic radiation source(34), such as a laser generating an irradiation beam to which thestained sperm are responsive. The electromagnetic radiation source (34)can be a laser operated at UV wavelength, such as at about 355 nm laseroperated at 175 mW. Alternatively, a laser can be run at a higher powerand split between several fluid streams.

The characteristics of individual sperm, particularly the presence of anX-chromosome or a Y-chromosome can be determined from the detectedfluorescence produced in response to the electromagnetic radiationsource (34). The DNA selective fluorescent dye binds stoichiometricallyto sperm DNA. Because X-chromosome bearing sperm contain more DNA thanY-chromosome bearing sperm, the X-chromosome bearing sperm can bind agreater amount of DNA selective fluorescent dye than Y-chromosomebearing sperm. Thus, by measuring the fluorescence emitted by the bounddye upon excitation, it is possible to differentiate between X-bearingspermatozoa and Y-bearing spermatozoa.

In order to achieve separation and isolation based upon stained spermcharacteristics, emitted light can be detected by the sensor (32) andthe information fed to an analyzer (36) coupled to a droplet chargerwhich differentially charges each drop (28) based upon thecharacteristics of the stained sperm contained within that drop (28). Inthis manner the analyzer (36) acts to permit the electrostaticdeflection plates (38) to deflect drops (28) based on whether or notthey contain the appropriate particle or cell.

As a result, the flow cytometer (10) acts to separate stained sperm bycausing the drops (28) containing sperm to be directed to one or morecollection containers (40). For example, when the analyzerdifferentiates sperm cells based upon a sperm cell characteristic, thedroplets entraining X-chromosome bearing spermatozoa can be chargedpositively and thus deflect in one direction, while the dropletsentraining Y-chromosome bearing spermatozoa can be charged negativelyand thus deflect the other way, and the wasted stream (that is dropletsthat do not entrain a particle or cell or entrain undesired orunsortable cells) can be left uncharged and thus is collected in anundeflected stream into a suction tube or the like as discussed in U.S.patent application Ser. No. 09/001,394, hereby incorporated by referenceherein. Naturally, numerous deflection trajectories can be establishedand collected simultaneously.

FIG. 3 illustrates comparisons of bivariate plots generated in sexsorting sperm which reflect the intensity of the excited DNA selectivefluorescent dye during sorting. Three bulls were stained with equalamounts of Hoechst 33342 and with an equal amount of yellow food dye No.6 and red food dye No. 40. The region labeled as R4 surrounds what isconsidered the “dead” sperm population, while the region R1 is centeredon viable, oriented sperm. It can be seen that for each bull yellow fooddye no. 6 provided superior quenching as for each bull the “dead”population, having the majority of the membrane comprised sperm, waspulled further away from the “live” population as compared to red fooddye No. 40.

FIG. 4 illustrates an example bivariate plot and univariate histogram ofthe parameters measured during sex selection by a FACS on a flowcytometer. The relative intensity of the live, or non-membranecompromised sperm can be seen on the bivatiate plot having a heightlabeled “B,” while the relative intensity of the quenched dead, orquenched membrane compromised sperm is labeled “A.” The differences inmagnitude of lines “A” and “B” demonstrate the degree to which amembrane compromised subpopulation of sperm is quenched and may berepresented as a fraction or a percentage. Without a dead quenching dye,both populations of sperm would overlap to a large extent, withunpredictable variations in the dead sperm.

The univarite histogram demonstrates the number of counts at particularvalues in the flow cytometer. Each peak represents one of eitherX-chromosome bearing sperm, or Y-chromosome bearing sperm, with someoverlap seen at the base. The amount of separation between the peaks canbe characterized in a number of ways. Here the line is indicated at “C”provides an indication of the peak height, whereas “D” provides anindication of the magnitude for the valley separating the peaks. High“D” values and low “C” values would indicate minimal overlap in thesubpopulations and excellent splits, whereas when the length of C and Dare equal, there is effectively no split. Split quality may becharacterized as a ratio of the distances indicated by C and D.

Example 1 Comparison of Dead Quenching Dyes for Post Thaw Motility SpermMotility in Bovine

Collection—

Sperm was collected from 10 different bulls on a routine collectionschedule using an artificial vagina over the course of 4 days. The 10bulls included both Jersey bulls and Holstein bulls. All ejaculatescontained greater than 60% progressive motility and greater than 70%morphological normal sperm. Antibiotics were added within 15 minutes ofcollection.

Staining—

Red food dye treatment 40% —A reduced volume of red food dye No. 40control was established. Sperm was diluted to 160×10⁶ sperm per ml in amodified TALP buffer, as described in Table 1, at a pH of 7.4. Samplescollected from Jersey bulls were incubated with 16 μl of Hoechst 33342per 2 ml of sample for between 45 and 60 minutes at 34° C., whilesamples collected from Holstein bulls were incubated 17 μl of Hoechst33342 per 2 ml of sample for between 45 and 60 minutes at 34° C. Afterincubation an equal volume of a second modified TALP was added reducingthe concentration to 80×10⁶. The second modified TALP includes thecomponents described in Table 1 with the addition of 4% egg yolk, 40 μMred food dye No. 40 (20 g/L) and the pH dropped to 5.5 with HCl.

Yellow Food dye treatment—Yellow food dye was included in a second groupat an equal concentration. Sperm was diluted to 160×10⁶ sperm per ml ina modified TALP buffer, as described in Table 1, at a pH of 7.4. Samplescollected from Jersey bulls were incubated with 14 μl of Hoechst 33342per 2 ml of sample for between 45 and 60 minutes at 34° C., while samplecollected from Holstein bulls were incubated 15 μl of Hoechst 33342 per2 ml of sample for between 45 and 60 minutes at 34° C. After incubationan equal volume of a second modified TALP was added reducing theconcentration to 80×10⁶. The second modified TALP includes thecomponents described in Table 1 with the addition 4% egg yolk, 20 g/Lyellow food dye No. 6 and a pH dropped to 5.5 with HCl.

Sorting—

Just prior to sorting, each sample was filtered though a 40 μm nylonmesh. Each sample was sorted with an MoFlo SX XDP (Beckman Coulter,Inc., CA USA) operating at 40 psi. A tris-citrate-fructose sheath fluidwas used comprising Tris(hydroxymethyl)aminomethane (Sigma Chemical Co,St. Louis, Mo., USA) citric acid monohydrate (Sigma Chemical Co, St.Louis, Mo., USA) and fructose (Sigma Chemical Co, St. Louis, Mo., USA).Antibiotics were additionally added to the sheath fluid.

Unquenched sperm having the desired sex characteristics were collectedinto a single tube with a tris-egg yolk catch fluid and the remainingsperm was removed with waste. The tris-egg yolk catch fluid comprised20% egg yolk, Tris(hydroxymethyl)aminomethane (Tris 200 mM; SigmaChemical Co, St. Louis, Mo., USA), citric acid monohydrate (65 mM; SigmaChemical Co, St. Louis, Mo., USA), fructose (56 mM; Sigma Chemical Co,St. Louis, Mo., USA) and antibiotics, where the sample remained for amaximum of approximately three hours.

Freezing—

Prior to freezing, the sorted sperm sample was cooled to about 5° C. andextended with a Tris-citrate-fructose solution similar to the catchfluid but additionally containing 12% glycerol. The cooled sorted samplewas centrifuged and resuspended in a Tris-citrate-fructose solution with20% egg yolk solution and 6% glycerol to arrive at a final concentrationof 10×10⁶ sperm/ml. Sperm was stored in divided into 2.1×10⁶ doses andstored in 0.25 ml straws which were then cryopreserved with liquidnitrogen vapor.

Results—

Straws were thawed the following day for motility measurements at 0 hourand 3 hours. Percent intact acrosomes were measured as well, and theoverall averages over all bulls on each day are illustrated in Table 3.Each bull tended to average higher post thaw motilies at 0 hour and at 3hours as well as improved acrosomal integrity.

TABLE 3 Post thaw Motility Bovine Motility % Average 0 Hour Red 56.6%Average 0 Hour Yellow 57.7% Average 3 Hour Red 47.0% Average 3 HourYellow 49.0% Average PIA Red 70.8% Average PIA Yellow 71.3%

Example 2 Comparison for Dead Quenching Dyes for Sorting Parameters inBovine

Collection—

8 Sperm collections were taken from 7 different bulls on a routinecollection schedule using an artificial vagina. The 7 bulls includedboth Jersey bulls and Holstein bulls. All ejaculates contained greaterthan 60% progressive motility and greater than 70% morphological normalsperm. Antibiotics were added within 15 minutes of collection. Portionsof each collected sample received three different dead quenching dyetreatments.

Staining—

Red food dye No. 40 treatment 100% (control)—A control was establishedwith a conventional volume of red food dye No. 40. Sperm was diluted to160×10⁶ sperm per ml in a modified TALP buffer, as described in Table 1,at a pH of 7.4. Samples collected from Jersey bulls were incubated with16 μl of Hoechst 33342 per 2 ml of sample for 60 minutes at 34° C.,while sample collected from Holstein bulls were incubated 17 μl ofHoechst 33342 per 2 ml of sample for between 45 and 60 minutes at 34° C.After incubation, an equal volume of a second modified TALP was addedreducing the concentration to 80×10⁶. The second modified TALP includesthe components described in Table 1 with the addition 4% egg yolk, 125μM red food dye No. 40 and a pH dropped to 5.5 with HCl.

Red food dye No. 40 treatment 40%—A second group was prepared in thesame way as the first, but with a 40% concentration of the red food dyeNo. 40, 50 μM red food dye No. 40.

Yellow food dye No. 6 treatment—Yellow food dye was included in thefinal group and was prepared in the same manner as Example 1. Theconcentration of yellow food dye No. 6 corresponds to the 40% red fooddye No. 40 treatment at a concentration of 50 μM.

Sorting—

Just prior to sorting, each sample was filtered though a 40 μm nylonmesh. Each sample was sorted with a MoFlo SX XDP (Beckman Coulter, Inc.,CA USA) operating at 40 psi. The sheath fluid described in Example 1 wasused for sorting.

The event rate was held as close to 40,000 per second as possible foreach sort and each sample was sorted for X-chromosome bearing sperm. Theeight sorts were carried out on 6 different machines in order tominimize instrumentation effects and to average out differences in eachinstruments performance due to nozzle, beam shaping, and drop drivefrequency.

Results—

In Tables 3-6 benefits to the sorting speed and sorting resolution aredemonstrated with a reduced concentration of yellow food dye No. 6.Sperm samples quenched with yellow food dye No. 6 are denoted Y6. Thecontrol amount of red food dye No. 40 is identified as R40-100%, andR40-40% represents a red food dye No. 40 at a 40% concentration ascompared to the control (125 μM vs. 50 μM).

Table 4 illustrates the average event rates across each sample anddemonstrates they were maintained within just over 1%. Table 5demonstrates a minor improvement in sort purity using a smaller amountof red food dye No. 40, and a further improvement in purity using yellowfood dye No. 6, with a corresponding reduction in DNA selectivefluorescent dye. Table 6 demonstrates a 14% increase in sort speed withthe yellow food dye No. 6 is used as a dead quenching dye. This is asignificant improvement given that the event rates were separated byonly about 1% and that the sort purities were also very similar. Table 7demonstrates that the X region contains 42.7% of the sperm population inthe samples quenched with yellow food dye No. 6, as opposed to 39.1% and38.4% for each of the red food dye quenchers. And this appears whereyellow food dye No. 6 out performs the red food dyes. The improvedresolution allows for excluding fewer cells which were not intended tobe excluded resulting in a larger percentage of cells in sort region,and faster sort times at similar event rates.

As the sorting region and sorting speeds improve, Table 8 illustratesthe number of coincident aborts also increase.

TABLE 4 AVERAGE EVENT RATE (events per second) R40- 100% 40412 R40- 100%40255 Y6 40675

TABLE 5 AVERAGE PURITY (percent) R40- 100% 89.5 R40- 100% 89.2 Y6 90.5

TABLE 6 AVERAGE SORT SPEED (sorts per second) R40- 100% 5406 R40- 100%5522 Y6 6194

TABLE 7 AVERAGE X REGION (percent) R40- 100% 38.4 R40- 100% 39.1 Y6 42.7

TABLE 8 AVERAGE ABORT RATE (aborts per second) R40- 100% 3310 R40- 100%3292 Y6 3540

Example 3 Comparison of Dead Quenching Dyes for Sperm Motility in Deerand Elk

Collection—

Each of deer and elk were collected in the Tris-egg yolk catch fluiddescribed as a catch media in Example 1, and shipped for sorting.

Staining—

A portion of each of the deer and the elk were stained by the red fooddye No. 40 treatment 40% with 16 μL Hoechst 33342 and the yellow fooddye No. 6 treatment with 14 μL Hoechst 33342 as described in Example 1.

Sorting and Freezing—

Samples were each bulk sorted with the same machine and parametersdescribed in Example 1. Both sorted X and sorted Y sperm were collectedinto a single catch. The sorted samples were then frozen like thosedescribed in Example 1.

Results—

Frozen thawed samples were then tested for post thaw motilities and postthaw DNA fragmentation. Post thaw motilities were slightly higher in Elksorted with a yellow quencher, while a more significant differenceexisted in progressive motility. In Table 10, post thaw DNAfragmentation in Elk can be seen at about double after 48 hours when redfood dye No. 40 is compared to yellow food dye No. 6 as a dead quencher.In Tables 9 and 11, Deer and Elk each demonstrated improved motility andprogressive motility in the samples quenched with yellow food dye No. 6and stained with 14 μL of Hoechst 33342 as compared to the red food dyeNo. 40 with 16 μL of Hoechst 33342.

TABLE 9 Post Thaw Motility Elk Motile Progressive RED TALP, 16st 0 HR51.0 29.0 Y6, 14st 55.5 38.0

TABLE 10 Post Thaw DNA fragmentation Elk Pre-sort Post Sort RED TALP,16st  0 HR 2.3 0.0 24 HR 5.6 0.3 48 HR 6 0.7 Y6, 14st  0 HR 2.3 0.0 24HR 3.3 0 48 HR 3.7 0.3

TABLE 11 Post Thaw Motility Deer Motile Progressive RED TALP, 16st 0 HR59.8 43.0 Y6, 14st 67.5 53.2

Example 4 Comparison of Dead Quenching Dyes for Sperm Motility in Dog

Collection—

Sperm was collected from a single canine at room temperature intoCaniPro Chill 5, available from Minitube, Wis., USA, then centrifugedinto a pellet and resuspended in a Tris buffer at a pH of 6.8.

Staining—

Samples were extended to 40×10⁶ sperm/ml in 1 ml volumes with the Trisbuffer described in Example 1 but set to a pH of 7.2. Ten samples werethen stained 4, 4, 5, 5, 7, 8, 8, 9, 9, and 10 μL of Hoechst 33342 andquenched with one of two quenching dyes at a concentration of 50 μM in asecond buffer Tris buffer.

Red food dye No. 40 quencher—1 ml Tris at a pH 6.3 was added with redfood dye No. 40 into samples stained with 4, 5, 8, 9, 10 μL Hoechst33342. The red food dye No. 40 was added at a concentration of about 50μM.

Yellow food dye No. 6 quencher—1 ml Tris at a pH 6.3 was added withyellow food dye No. 6 into samples stained with 4, 5, 7, 8, 9 μL Hoechst33342. The yellow food dye No. 6 was added at the same concentration asthe red food dye No. 40 quencher, at about 50 μM.After quenching, each sample had a total volume of 2 mL.

Sorting—

5 million cells were sex sorted with a MoFlo SX XPD into 50 mL Falcontubes having 3.5 mL of a Tris-egg yolk catch fluid, described in Example1 for both X and Y populations. Two samples were sorted, the red fooddye No. 40 quenched treatment with 9 μL Hoechst 33342 and the yellowfood dye No. 40 quenched treatment with 7 μL Hoechst 33342.

On the sorter, the samples sorted with red quenched treatment with 9 μLHoechst 33342 demonstrated 50.73 percent of sperm in the X region and8.92 percent were gated as dead. Samples sorted with the yellow quencherhad 48.7 percent of sperm in the X region and only 4.42 percent of thesperm gated as dead.

Freezing—

Straws were then frozen and stored in liquid nitrogen.

Thawing—

Straws were thawed for 30 sec in a 38° C. water bath and theirmotilities were examined on CASA.

Results—

Table 12 illustrates the average motilities and progressive motilitiesfor the sorted treatments. Canine sperm quenched with yellow food dyeNo. 6 provided about 4.5% more motile sperm in the X population and 6.5%more motile sperm in the Y population. Similarly, the yellow quencheddye provided 7% better progressive motility in the X population and 8%better progressive motility in the Y population.

TABLE 12 Dead quenching dye motilities TIME Total cells MotileProgressive X YELLOW 6 AVERAGE 0 Hr 192 24 18.5 Y YELLOW 6 AVERAGE 0 Hr322 25 21 X RED TALP AVERAGE 0 Hr 312 19.5 11.5 Y RED TALP AVERAGE 0 Hr328 18.5 13

Example 5 Comparison of Dead Quenching Dye Amounts Vs Hoechst 33342Stain Amounts

Collection—

A single Jersey bull was collected at an initial sperm concentration1900×10⁶ sperm/ml in a volume of 5 ml. The progressive motility of thesperm was 65% with normal morphologies greater than 65%.

Staining—

The entire ejaculate of sperm was suspended in first staining buffer,specifically a 7.4 pH TALP described in TABLE 1, to a concentration of160×10⁶ sperm per mL (determined on nucleocounter) and divided intothree aliquots of about 20 mL into 50 mL Falcon tubes and pre-warmed for10 minutes at 34° C. The DNA selective fluorescent dye, Hoechst 33342,was added and samples were held at 34° C. for 60 minutes. 3 differentHoechst 33342 Stain Levels were used=12 μL, 14 μL and 16 μL per 2 mL(effective concentrations of 48 μM, 56 μM, 64 μM).

Preparation of the second buffer including the dead quenching dye—A 2 Lbag of TALP, described in TABLE 1, was adjusted to pH 5.60 using HCl andthen sterile filtered. (Note: This test did not use 0.3% v/v BSA or 4%v/v Egg Yolk in the second buffer). For each of the four dyes tested(R40—red food dye No. 40, Y5—yellow food dye No. 5, Y6—yellow food dyeNo. 6 and PR—phenol red), an appropriate number of milligrams of eachpowder was placed in a Erlenmeyer Flask and then an exact amount of TALPpH 5.60 was added to create a 250 μM concentration of the dye. Theresult was a working TALP stock for each dye with a 100% (125 μM)relative amount of dye 50% relative (62.5 μM) and 20% relative (25 μM)solutions were made by dilution of 100% by TALP pH 5.60.

Controls—

Single tubes of stained sperm at each of the three Hoechst 33342concentrations were treated with TALP pH 5.60 which had no deadquenching dye present. Single tubes using Vitamin B12 (red coloredchemical) with stock concentration of 15 μM (12% relative) were tested.

Sorting and Data Acquisition—

After this “bulk” staining for 60 minutes, 1 mL aliquots of stainedsperm were combined with 1 mL aliquots of the second buffer includingthe dead quenching dyes. Samples sat for approximately 30 minutes atroom temperature before being analyzed on a MoFlo SX (Beckman Coulter,USA) sperm sorting flow cytometer. Event rates were held as close to13,000 as possible at the point of data acquisition.

The total number of individual treatments was 42 tubes, each containing2 mL of stained sample. The alignment of the sorter was establishedusing nuclei and confirmed with live sample from the 50% Red 40 series.Once good alignment was established, each tube was placed on the sorterlong enough to establish a stabile dead gate population (about 30seconds) and then a screenshot was taken and saved as a .jpeg image.This approach of rapidly documenting each sample is used to assure themost consistent alignment between all samples. Realignment of sorterwill not cause differences in the intensities of live and dead gatepopulations but will cause differences in split quality.

Results—

FIG. 4 illustrates an example histogram. The ratio of dead gateintensity divided by live gate intensity is created by dividing thedistance “A” by the distance “B” shown as a percentage. Lower valuesmean higher levels of quenching. The ratio of peak height divided byvalley height is created by dividing the distance “C” by the distance“D”. A value of 1.00 means “no split” while higher values mean “bettersplits”.

TABLE 13 shows the ratios created by making this measurement on each of42 histograms generated in the data acquisition described above. 16 μLdata was omitted in the split data of TABLE 13 because there was mostlyno split in nearly all samples (due to “overstained condition”). FIG. 5shows the average data for dead gate intensity divided by live orientedgate intensity. FIG. 6 illustrates the average data for 12 μL and 14 μLstain series for the height of peak divided by height of valley.

From the dead and live intensity data, the absorption of the four dyes(ability to create a dead gate population) is ordered as red food dyeNo. 40 and yellow food dye No. 6 being about equal and better than theremaining dyes. Of the remaining dyes yellow food dye No. 5 had betterquenching properties than phenol red. From the split quality data, fooddye improves the quality of split compared to no food dye control.Relative level of 20% food dye might be better than 40%.

TABLE 13A dead quenching and split enhancing capacities Hoechst No stainRed 40 Yellow 6 μL/2 mL 0 125 μM 62.5 μM 25 μM 125 μM 62.5 μM 25 μM DeadIntensity 16 100% 35% 50% 65% 40% 52% 70% 14 100% 41% 53% 72% 40% 52%74% 12 100% 37% 53% 68% 37% 50% 68% Average 100% 38% 52% 68% 39% 51% 71%Height of Peak/Height of Valley 16 1.00 1.21 1.00 1.00 1.26 1.00 1.13 141.44 2.00 1.62 1.94 1.80 1.62 2.07 12 1.50 1.75 1.65 2.05 1.73 1.70 2.05Average 1.47 1.88 1.64 2.00 1.77 1.66 2.06

TABLE 13B dead quenching and split enhancing capacities Hoechst Yellow 5Phenol Red B12 μL/2 mL 125 μM 62.5 μM 25 μM 125 μM 62.5 μM 25 μM 0.25Dead Intensity 16 55% 70% 90% 78% 80% 90% 100% 14 63% 77% 90% 80% 90%97% 100% 12 54% 74% 90% 80% 90% 94% 100% Average 57% 74% 90% 79% 87% 94%100% Height of Peak/Height of Valley 16 1.03 1.00 1.00 1.00 1.00 1.001.00 14 1.92 1.70 1.54 1.75 1.87 1.65 1.62 12 1.72 1.60 1.90 1.49 1.621.82 1.61 Average 1.82 1.65 1.72 1.62 1.75 1.74 1.62

Example 6 Comparison of Dead Quenching Dye for Yellow Food Dye No. 6 andPhenol Red

Collection—

A single Jersey bull was collected at an initial sperm concentration1810×10⁶ sperm/ml in a volume of 3.9 ml. The progressive motility of thesperm was 65% with normal morphologies greater than 50%.

Staining—

Sperm were suspended at 320 million per mL in 30 mL a first buffer,(TALP described in table 1 at a pH 7.4) with 12 μL of Hoechst 33342 per2 mL (48 μM) of stain and incubated for 1 hour at 34° C. Stained spermwas then placed as 1 mL aliquots in sample tubes and a 1 mL volume ofthe appropriate TALP was added. The same second buffer was used asexample 1, namely a modified 5.60 pH TALP. Since the color, and hencethe absorption characteristics of phenol red is pH sensitive, it wasmade in a first series with TALP pH 5.60 resulting in a pH of 6.70 (likeexample 1) and in a second series the second buffer had a pH of 7.40(the same buffer as the first buffer, with the addition of the quenchingdyes).

Sorting and Data Acquisition—

Each of the stained samples was sorted on a MoFlo SX (Beckman Coulter,USA) as described in Example 6. Screen shots were taken and the deadgate intensity and the split quality were measured in the same waydescribed in Example 1 and recorded in TABLE 14.

TABLE 14 Phenol Red Yellow 5 Red 40 1250 μM 875 μM 750 μM 500 μM 250 μM125 μM 62.5 μM Intensity <30% <30% <30% 43% 48% 63% 53% PV 1.00 1.001.00 1.00 1.00 1.00 1.00 Phenol Red (pH 6.70) Yellow 6 1250 μM 875 μM750 μM 500 μM 250 μM 125 μM 62.5 μM Intensity 41% 45% 55% 67% 70% 79%50% PV 1.29 1.20 1.25 1.25 1.17 1.00 1.00 Phenol Red (pH 7.40) 1250 μM875 μM 750 μM 500 μM 250 μM 125 μM Intensity 43% 47% 56% 66% 76% 85% PV1.30 1.44 1.46 1.51 1.22 1.27

Results—

From the additional data on the proportional intensity of the dead gateit can be seen that it takes about five times as much yellow food dyeNo. 5 to get a similar dead gate intensity ratio as for red food dye No.40 and yellow food dye No. 6. Furthermore, phenol red at both of the pHsettings tested, is far less capable of quenching to create a dead gate,requiring twelve times more than red food dye No. 40 and yellow food dyeNo. 6. With respect to the ability to efficiently make a deadpopulation, phenol red does not appear to be a good candidate, comparedto red food dye No. 40 and yellow food dye No. 6 or yellow food dye No.5. FIG. 7A illustrates a comparison of the concentrations required toachieve dead gates with each of the tested dye, combining the date fromexample 6 and example 7. It can be seen yellow food dye No. 6 and redfood dye No. 40 perform similarly with respect to creating a dead gate,but that yellow food dye No. 5 and phenol red require significantlyhigher concentrations to affect a gate dead, or membrane compromisedsperm.

Nonetheless, phenol red was the only of these four dyes which created asplit improvement in this sample. This sample was “understained” at 12μL/2 mL, meaning that there was either not enough Hoechst 33342 foruniform staining. Because of this, the control samples did not have asplit. It was very surprising, therefore, to see splits emerge in thepresence of phenol red. Also, the quality of the split was improved bythe higher pH TALP. At basic pH, phenol red (pKa 8.0) turn to an ionicalkaline form and exhibits a pink-red color.

Keeping in mind a value of 1.00, is the equivalent of no split, the datain FIG. 7B is surprising in that splits emerge at all testedconcentrations of phenol red. It appears that, especially at the higherpH configuration, phenol red acts to reduce noise and may be able toperform as a split enhancing dye.

Example 7 The Combined Effects of an Improved Dead Quenching Dye and aSplit Enhancing Dye

Collection—

A single Jersey bull was collected at an initial sperm concentration1810×10⁶ sperm/ml in a volume of 3.9 ml. The progressive motility of thesperm was 65% with normal morphologies greater than 50%.

Staining—

Sperm were stained in previously described first buffer TALP (pH 7.4)(described in TABLE 1 and in Example 1) at 160 million sperm/mL with theaddition of 48 μM Hoechst 33343 (12 μL/2 mL) and 62 μM yellow food dyeNo. 6 and incubated for 1 Hour at 34° C. The sperm sample was thenconcentrated by centrifugation and resuspended at concentrations of 100,150, 250, and 300 million sperm per mL, respectively. Samples were splitinto two equal aliquots and in one of each pair of samples, a stocksolution of phenol red was added to make a final phenol redconcentration of 620 μM. Samples at each concentration were preparedwith the dead quencher yellow food dye No. 6 at a concentration of 62μM. At each concentration, one control was compared to the addition ofphenol red as a split enhancing dye at a concentration of 620 μM.

Sorting and Data Acquisition—

Sorting was performed on a MoFlo SX (Beckman Coulter, USA) insubstantially the same manner described in Example 5. After calibration,screen shots of sorting histograms were taken and split quality wasmeasured by the peak to valley ration in the same way described inExample 5. The resulting data is found in Table 15.

TABLE 15 Phenol Red Concentration Concentration Concentration Height ofPeak of Cells of Yellow 6 of Phenol Red Divided by (million per mL) (μM)(μM) Height of Valley 100 62 0 1.26 100 62 620 1.49 150 62 0 1.30 150 62620 1.34 250 62 0 1.25 250 62 620 1.43 300 62 0 1.23 300 62 620 1.36

Results—

At each sperm concentration tested the combination of the splitenhancing dye, phenol red, with the dead quenching dye, yellow food dyeNo. 6, gives better peak quality than the dead quencher at theconcentrations of yellow food dye No. 6 tested.

Example 8 Phenol Red as an Added Split Enhancing Dye

Collection—

Two bulls were collected for the Example 8. The Bull 1 was a Jerseybulls and Bull 2 was a Holstein collected substantially in the mannerdescribed in example 1.

Staining—

Sperm from Bull 1 extended to 160 million per with a first buffer, (TALPdescribed in table 1 at a pH 7.4) and divided into two groups forstaining. The first group was stained with 44 μM Hoechst 33342 and thesecond group was stained with 60 μM Hoechst 33342. Similarly, bull 2 wassuspended to 160 million per mL the first buffer, (TALP described intable 1 at a pH 7.4) and divided into three groups for staining. Thefirst group was stained with 28 μM Hoechst 33342 and the second groupwas stained with 44 μM Hoechst 33342 and the third group was stainedwith 66 μM Hoechst 33342. Each group was incubated for 1 hour at 34° C.and then extended in an equal volume of the second buffer. The samesecond buffer was used as Example 1, namely a modified 5.60 pH TALP.

The stained samples were each treated with a variety of quenching dyesand combinations of quenching dyes indicated in below. Table 16 providesparings of results where phenol red is investigated in severalconcentrations, alone and in combination with red food dye No. 40 andyellow food dye No. 6, for comparison against a control lacking only thephenol red.

Sorting and Data Acquisition—

Sorting was performed on a MoFlo SX (Beckman Coulter, USA) insubstantially the same manner described in Example 5. After calibration,screen shots of sorting histograms were taken and histograms splitquality was measured as a peak to valley ratio in the same way describedin Example 5 with reference to FIG. 4 and the resulting data is found inTable 16.

Results—

At each staining level of the DNA selective fluorescent dye, with bothdead quenching dyes (Red 40 and Yellow 6), the addition of phenol redalways improved split quality.

TABLE 16 Phenol Red as a split enhancing dye Conc Conc Conc Conc Hoechst33342 R40 Y6 PR Bull (μM) (μM) (μM) (μM) PV Bull 1 44 0 0 0 1.44 Bull 144 0 0 500 1.97 Bull 1 44 0 50 0 1.20 Bull 1 44 0 50 250 1.52 Bull 1 600 0 0 1.60 Bull 1 60 0 0 375 2.38 Bull 1 60 20 0 0 1.62 Bull 1 60 20 01200 2.12 Bull 1 60 0 50 0 1.39 Bull 1 60 0 50 375 2.38 Bull 2 28 0 0 01.63 Bull 2 28 0 0 375 1.96 Bull 2 28 0 50 0 1.64 Bull 2 28 0 50 3752.05 Bull 2 44 20 0 0 1.54 Bull 2 44 20 0 375 1.64 Bull 2 44 0 50 0 1.35Bull 2 44 0 50 225 1.56 Bull 2 44 0 50 375 1.75 Bull 2 60 0 50 0 1.35Bull 2 60 0 50 375 1.69 Bull 2 60 0 375 0 1.72 Bull 2 60 0 375 375 2.31

Example 9 Red Food Dye No. 3 as an Added Split Enhancing Dye

Collection—

A single bull was collected for the Example 9 in the same way describedin Example 5.

Staining—

Sperm from Bull 1 extended to 160 million per with a first buffer, (TALPdescribed in table 1 at a pH 7.4) and stained with 60 μM Hoechst 33342,as described in Example 1 at 34° C. for an hour. Each sample was thenextended in an equal volume the second buffer, 5.6 pH modified TALPdescribed in Example 5.

The second buffer also included either the dead quenching dye yellowfood dye No. 6, or red food dye No. 40 at a concentration of 60 μM. Thesecond buffer was additionally treated with concentrations of 25, 50,100, 150, 300, or 600 μM of red food dye No. 3, as indicated in Table 6.

Sorting and Data Acquisition—

Sorting was performed on a MoFlo SX (Beckman Coulter, USA) insubstantially the same manner described in Example 6. After calibration,screen shots of sorting histograms were taken and the split quality wasmeasured in terms of peak to valley ratios in the same way described inExample 5 with reference to FIG. 4 and the resulting data is found inTable 17.

Results—

For each sample tested in Table 17, increasing concentrations or redfood dye No. 3 improved the split quality up to the concentration of 300μM. At a concentration of 600 μM the split enhancer red food dye No. 3still provided improved splits compared to the control (0 red food dyeNo. 3), but not compared to a concentration of 300 μM.

TABLE 17 Red food dye No. 3 as a split enhancing dye Conc Conc Conc ConcHoechst 33342 R40 Y6 R3 Bull (μM) (μM) (μM) (μM) PV Bull 3 60 0 50 01.26 Bull 3 60 0 50 25 1.45 Bull 3 60 0 50 100 1.38 Bull 3 60 0 50 1501.41 Bull 3 60 0 50 300 1.66 Bull 3 60 0 50 600 1.31 Bull 3 60 0 50 01.32 Bull 3 60 0 50 50 1.33 Bull 3 60 0 50 100 1.55 Bull 3 60 0 50 3001.68 Bull 3 60 0 50 600 1.42 Bull 3 60 50 0 0 1.44 Bull 3 60 50 0 501.63 Bull 3 60 50 0 100 1.65 Bull 3 60 50 0 200 1.78 Bull 3 60 50 0 3002.15

Example 10 Split Enhancing Dyes at Various Concentrations

Collection

Eight bulls were collected, including 4 Jersey bulls, 3 Holstein bullsand one mixed dairy-breed. The ejaculates were collected atconcentrations ranging from 1700×10⁶ sperm/ml to 1100×10⁶ sperm/ml andall had progressive motility of a least 60%.

Staining—

Sperm from each bull was extended to 160 million per ml with a firstbuffer, (TALP described in table 1 at a pH 7.4) and stained with 64 μM(16 μL/2 mL) Hoechst 33342 at 34° C. Additionally, Phenol Red, Red FoodDye No. 2, Red Food Dye No. 3, and Red Food Dye No. 4, were each testedas split enhancing dyes in the first buffer at concentrations of 15 μM,45 μM and 125 μM. For each bull, each concentration of each dye wasincubated for periods of 30 minutes, 45 minutes, 60 minutes, and 75minutes were utilized before a second buffer with an additional dye wasapplied.

Each sample was then extended in an equal volume of a second bufferwhich comprised a 5.5 pH modified TALP having 25 μM yellow food dye No.6 (acting as a dead quenching dye) in addition to the split enhancingdye provided in the first buffer. The second buffer was applied to everysample at 30 minutes, 45 minutes, 60 minutes, and 75 minutes. A controlwas additionally taken without any split enhancing dye for every bull atevery time interval.

Sorting and Data Acquisition—

Sorting was performed on a MoFlo SX (Beckman Coulter, USA) insubstantially the same manner described in Example 5. After calibration,screen shots of sorting histograms were taken and split quality wasmeasured by peak to valley ratios in the same way described in Example 5with reference to FIG. 4 and the resulting data, averaged over all 8bulls, is found in Tables 18-21. Additionally, screen shots of bivariateplots were taken to measure the added dead quenching capacity of themixed dead quenching and split enhancing dyes.

Results—

Referring to Table 18, red food dye No. 2 did not provide a splitenhancing, or noise reducing effect. Instead, averaged over the 8 bulls,every concentration of Red Food dye no. 2 resulted in a reduced peak tovalley ratio, as compared to a control. Red food dye No. 2 did, however,provide a strong dead quenching capacity as seen in FIG. 8.

Referring to Table 19, red food dye No. 3, on average, provided initialsplit enhancements at 15 μM and 45 μM, but the control was as good as 45μM after 60 minutes and as good as 15 μM after 75 minutes.

Referring to Table 20, Phenol Red provided consistent split improvementsat all concentrations after 30 minutes. This improvement may provide ameans for shortening incubation times, or may simply provide for higherthroughput and higher purity in sex sorting protocols.

Referring to Table 21, red food dye No. 4 provides distinct improvementsat all concentrations and at all times. Referring to FIG. 9, the splitenhancement can be at each of 15 μM, 45 μM and 125 μM, particularly at45 minutes and beyond. Red Food dye no. 4 may provide noise reductionand split enhancement at lower concentrations approaching 0 μM and doesnot show any sign of tailing off at the top end of the range tested. Redfood dye No. 4 provided more consistent improvements acrossconcentrations and times than phenol red and it is believed may continueto provide benefits in the ranges in which Phenol Red was tested inExample 9, such as concentrations as high as 1200 μM.

At the highest tested concentration red food dye No. 4 additionallyprovided a significant increase in quenching the dead subpopulation. Redfood dye No. 4 may provide for a robust split enhancing dye over a largerange of concentrations, and more provide the added benefit of reducingthe amount of dead quenching dye required for sorting sperm.

TABLE 18 Red Food Dye No. 2 - Average 8 bulls 30 Min 45 Min 60 Min 75Min Control- 0 μM Red 2 1.07 1.23 1.58 1.80 15 μM Red 2 1.06 1.15 1.541.73 45 μM Red 2 1.00 1.05 1.12 1.38 125 μM Red 2 1.00 1.00 1.00 1.00

TABLE 19 Red Food Dye No. 3 - Average 8 bulls 30 Min 45 Min 60 Min 75Min Control- 0 μM Red 3 1.07 1.23 1.58 1.80 15 μM Red 3 1.05 1.43 1.781.79 45 μM Red 3 1.05 1.36 1.58 1.60 125 μM Red 3 1.00 1.02 1.10 1.32

TABLE 20 Phenol Red - Average 8 bulls 30 Min 45 Min 60 Min 75 MinControl- 0 μM Red 3 1.07 1.23 1.58 1.80 15 μM Red 3 1.14 1.30 1.77 1.8745 μM Red 3 1.01 1.36 1.68 1.81 125 μM Red 3 1.08 1.42 1.73 1.84

TABLE 21 Red Food Dye No. 4 - Average 8 bulls 30 Min 45 Min 60 Min 75Min Control- 0 μM Red 4 1.07 1.23 1.58 1.80 15 μM Red 4 1.09 1.44 1.791.91 45 μM Red 4 1.18 1.50 1.76 1.94 125 μM Red 4 1.13 1.45 1.81 1.95

As can be easily understood from the foregoing, the basic concepts ofthe present invention may be embodied in a variety of ways. Theinvention involves numerous and varied embodiments of staining sperm forsex sorting including, but not limited to, the best mode of theinvention.

As such, the particular embodiments or elements of the inventiondisclosed by the description or shown in the figures or tablesaccompanying this application are not intended to be limiting, butrather exemplary of the numerous and varied embodiments genericallyencompassed by the invention or equivalents encompassed with respect toany particular element thereof. In addition, the specific description ofa single embodiment or element of the invention may not explicitlydescribe all embodiments or elements possible; many alternatives areimplicitly disclosed by the description and figures.

It should be understood that each element of an apparatus or each stepof a method may be described by an apparatus term or method term. Suchterms can be substituted where desired to make explicit the implicitlybroad coverage to which this invention is entitled. As but one example,it should be understood that all steps of a method may be disclosed asan action, a means for taking that action, or as an element which causesthat action. Similarly, each element of an apparatus may be disclosed asthe physical element or the action which that physical elementfacilitates. As but one example, the disclosure of “sorter” should beunderstood to encompass disclosure of the act of “sorting”—whetherexplicitly discussed or not—and, conversely, were there effectivelydisclosure of the act of “sorting”, such a disclosure should beunderstood to encompass disclosure of a “sorter” and even a “means forsorting.” Such alternative terms for each element or step are to beunderstood to be explicitly included in the description.

In addition, as to each term used it should be understood that unlessits utilization in this application is inconsistent with suchinterpretation, common dictionary definitions should be understood to beincluded in the description for each term as contained in the RandomHouse Webster's Unabridged Dictionary, second edition, each definitionhereby incorporated by reference.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more of that entity. As such, the terms “a”or “an”, “one or more” and “at least one” can be used interchangeablyherein.

All numeric values herein are assumed to be modified by the term“about”, whether or not explicitly indicated. For the purposes of thepresent invention, ranges may be expressed as from “about” oneparticular value to “about” another particular value. When such a rangeis expressed, another embodiment includes from the one particular valueto the other particular value. The recitation of numerical ranges byendpoints includes all the numeric values subsumed within that range. Anumerical range of one to five includes for example the numeric values1, 1.5, 2, 2.75, 3, 3.80, 4, 5, and so forth. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. When a value is expressed as an approximation by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment.

The background section of this patent application provides a statementof the field of endeavor to which the invention pertains. This sectionmay also incorporate or contain paraphrasing of certain United Statespatents, patent applications, publications, or subject matter of theclaimed invention useful in relating information, problems, or concernsabout the state of technology to which the invention is drawn toward. Itis not intended that any United States patent, patent application,publication, statement or other information cited or incorporated hereinbe interpreted, construed or deemed to be admitted as prior art withrespect to the invention.

The claims set forth in this specification, if any, are herebyincorporated by reference as part of this description of the invention,and the applicant expressly reserves the right to use all of or aportion of such incorporated content of such claims as additionaldescription to support any of or all of the claims or any element orcomponent thereof, and the applicant further expressly reserves theright to move any portion of or all of the incorporated content of suchclaims or any element or component thereof from the description into theclaims or vice versa as necessary to define the matter for whichprotection is sought by this application or by any subsequentapplication or continuation, division, or continuation-in-partapplication thereof, or to obtain any benefit of, reduction in feespursuant to, or to comply with the patent laws, rules, or regulations ofany country or treaty, and such content incorporated by reference shallsurvive during the entire pendency of this application including anysubsequent continuation, division, or continuation-in-part applicationthereof or any reissue or extension thereon.

We claim:
 1. A method of gender enriching one or more spermsubpopulations comprising the steps of: a. obtaining a population ofsperm having both X-chromosome bearing sperm and Y-chromosome bearingsperm; b. staining at least a portion of the population of sperm with afirst dye that is a DNA selective fluorescent dye which fluoresces whenexcited; c. staining said portion of the population of sperm with asecond dye that is a dead quenching dye for selectively quenchingfluorescence emitted by the DNA selective fluorescent dye withinmembrane compromised sperm; d. staining said portion of the populationof sperm with a third dye that is a split enhancing dye; and e. sortingthe stained sperm into one or more gender enriched subpopulations ofviable sperm.
 2. The method of claim 1 wherein the dead quenching dye isimpedance matched to the DNA selective fluorescent dye.
 3. The method ofclaim 1 wherein the dead quenching dye is a yellow, orange, ororangish-red dye.
 4. The method of claim 1 wherein the dead quenchingdye is selected from yellow food dye No. 6, yellow food dye No. 5 andcombinations thereof.
 5. The method of claim 1 wherein the deadquenching dye comprises yellow food dye No. 6 in a concentration between12.5 μM and 250 μM.
 6. The method of claim 1 wherein the dead quenchingdye comprises yellow food dye No. 6 in a concentration between 25 μM and125 μM.
 7. The method claim 1 wherein the DNA selective fluorescent dye,when excited by electromagnetic radiation, differentially fluoresces inX-chromosome bearing sperm and in Y-chromosome bearing sperm.
 8. Themethod of claim 7 wherein the dead quenching dye differentiallypermeates membrane compromised sperm so that unquenched sperm areenriched with respect to sperm viability compared to the population ofsperm.
 9. The method of claim 8 wherein the dead quenching dyedifferentially permeates membrane compromised sperm so that at least amajority of dead sperm are quenched by the dead quenching dye.
 10. Themethod of claim 1 wherein the DNA selective fluorescent dye is selectedfrom the group consisting of: UV excitable dyes, visible light excitabledyes, fluorescent polyamides, fluorescent nucleotide sequences, sexspecific antibodies, and combinations thereof.
 11. The method of claim10 wherein the DNA selective fluorescent dye comprises Hoechst 33342.12. The method of claim 11 wherein the step of staining the populationof sperm further comprises the step of incubating the population ofsperm with a concentration of Hoechst 33342 less than 42 micromolar. 13.The method of claim 1 wherein the step of obtaining a population ofsperm further comprises one selected from: obtaining neat semen;obtaining extended semen, obtaining frozen thawed sperm, poolingextended semen, and combinations thereof.
 14. The method of claim 1wherein the step of staining the sperm with a DNA selective dye furthercomprises the step of diluting the population of sperm with a firststaining buffer including the DNA selective fluorescent dye for anincubation period of between about 20 minutes and about an hour and ahalf.
 15. The method of claim 14 wherein the step of staining saidportion of the population of sperm with a quenching dye furthercomprises the step of diluting the sperm population with a secondstaining buffer including the dead quenching dye and the split enhancingdye after the incubation period.
 16. The method of claim 14 wherein thestep of staining said portion of the population of sperm with aquenching dye further comprises the step of diluting the spermpopulation with a second staining buffer after the incubation period,wherein the second staining buffer includes the dead quenching dye andfirst staining buffer includes the split enhancing dye.
 17. The methodof claim 14 or wherein the dead quenching dye and the split enhancingdye are contained in the first staining buffer.
 18. The method of claim1 wherein the step of staining the sperm with a DNA selective dyefurther comprises the step of diluting the population of sperm with afirst staining buffer including the DNA selective fluorescent dye for anincubation period of between about 30 minutes and about 75 minutes. 19.The method of claim 1 wherein the split enhancing dye further comprisesa dye having a local maximum absorption wave length greater than about520 nm.
 20. The method of claim 19 wherein the local maximum absorptionwave length of the split enhancing dye is between about 520 nm and about600 nm.
 21. The method of claim 1 wherein the split enhancing dyecomprises a red dye.
 22. The method of claim 21 wherein the splitenhancing dye comprises a dye selected from the group consisting of:phenol red, red food dye No. 4, red food dye No. 3, red food dye No. 2,and combinations thereof.
 23. The method of claim 22 wherein the splitenhancing dye is phenol red added in a concentration between about 0.1μM and about 1200 μM.
 24. The method of claim 22 wherein the splitenhancing dye is phenol red added in a concentration between about 5 μMand about 375 μM.
 25. The method of claim 22 wherein the split enhancingdye is red food dye No. 4 added in a concentration between about 0.1 μMand about 1200 μM.
 26. The method of claim 22 wherein the splitenhancing dye is red food dye No. 4 added in a concentration betweenabout 5 μM and about 375 μM.
 27. The method of claim 22 wherein thesplit enhancing dye is red food dye No. 4 added in a concentrationbetween about 15 μM and about 125 μM.
 28. The method of claim 1 whereinthe dead quenching dye and the split enhancing dye are added in a singlestaining treatment.
 29. The method of claim 1 wherein the dead quenchingdye is yellow food dye no. 6 and the split enhancing dye is red food dyeno.
 4. 30. The method of claim 1 wherein the local maximum absorptionwave length of the dead quenching dye is between about 420 nm and about500 nm and wherein the local maximum absorption wave length of the splitenhancing dye is between about 520 nm and about 600 nm.
 31. The methodof claim 1 wherein the step of sorting further comprises the steps of:a. establishing a fluid stream; b. introducing the stained sperm intothe fluid stream; c. exciting DNA selective fluorescent dye associatedwith the DNA of the stained sperm; d. detecting the fluorescenceemission produced by the stained sperm when excited; e. differentiatingquenched and unquenched sperm sub-populations within the stained sperm;f. differentiating X-chromosome bearing sperm and Y-chromosome bearingsperm in the unquenched sperm subpopulation based upon the detectedfluorescence of the unquenched sperm; and g. selecting sperm to form atleast one gender enriched subpopulation of viable sperm.
 32. The methodof claim 31 wherein the step of sorting is performed with a flowcytometer.
 33. The method of claim 31 wherein the step of exciting DNAselective fluorescent dye associated with the DNA of the stained spermfurther comprising interrogating the stained sperm with a laseroperating at a UV wavelength.
 34. The method of claim 32 wherein theflow cytometer is selected from a jet-in-air flow cytometer and a closedchamber flow cytometer.
 35. The method of claim 1 wherein the step ofsorting is performed on a microfluidic chip.